α7 nAChRs belong to the ligand-gated ion channel superfamily of Cys-loop receptors. The Cys-loop superfamily includes muscle and neuronal nAChRs, 5-hydroxytryptamine type 3 (5HT3), γ-aminobutyric acidA (GABAA), GABAC and glycine receptors. α7 nAChRs are allosteric proteins which recognize acetylcholine and choline as the orthosteric ligand and bind nicotine at the orthosteric site. Neuronal α7 nAChRs contain 5 orthosteric sites per receptor. Agonist binding to the orthosteric site transmits an allosteric effect which modulates the functional states of the receptor depending on the concentration and kinetics of agonist application. Four functional states have been described for nAChRs: one open and three closed states (resting, fast-onset desensitized, slow-onset desensitized). Activation of neuronal nAChRs mediates fast synaptic transmission and controls synaptic transmission by the major inhibitory and excitatory neurotransmitters, GABA and glutamate.
α7 nAChRs mediate the predominant nicotinic current in hippocampal neurons. The α7 nAChR was initially identified from a chick brain library as an α-bungarotoxin binding protein that exhibits ˜40% sequence homology to other nAChRs. α7 nAChRs share similar features of other neuronal and muscle nAChRs such as a pentameric Cys-loop receptor structure and M2 segment of each subunit lining of the channel pore, however the α7 nAChR exhibits a homopentameric structure when reconstituted in Xenopus oocytes, a characteristic shared only with the α8 and α9 nAChRs. Heterologously expressed homomeric α7 nAChRs in Xenopus oocytes are inactivated by α-bungarotoxin with high affinity, whereas other nAChRs are not. α7 nAChRs have also been pharmacologically identified by distinct types of whole cell currents elicited by nicotinic agonists in hippocampal neurons. When exposed to various nicotinic agonists whole cell recordings from cultured hippocampal neurons show, in general, type IA currents that have a very brief open time, high conductance, very high Ca++ permeability, rapid decay, and are sensitive to blockade by MLA and α-bungarotoxin. The properties of these nicotinic currents in hippocampal neurons correspond to the currents mediated by α7 nAChRs expressed in oocytes. We are specifically interested in α7 nAChRs because of their role in regulating fast synaptic transmission in the hippocampus where it provides a specific target for the modulation of hippocampal function.
GABAA receptors that contain α5 subunits show distinct immunocytochemical, mRNA hybridization, and selective radioligand binding patterns that are specific to hippocampal structures in mammalian brain. Immunoprecipitated GABAA α subunits from the hippocampus, but not cortex or whole rat brain, show α5 immunoreactivity. Furthermore tonic inhibition of CA1 pyramidal cells in the hippocampus is mediated, in part, by GABAA α5 receptors. Genetic alteration of GABAA α5 receptors causes behavioral responses consistent with enhanced hippocampal-dependent learning and memory such as spatial learning and associative learning. A series of triazolophthalazines with selective negative allosteric modulation of GABAA α5 receptors are reported to be efficacious in the delayed matching to position test in the water maze, a hippocampal-dependent animal cognition model (Dawson et al. J. Pharmacol. Exp. Ther. 316: 1335-1345, 2006). Therefore GABAA α5 receptors may provide a suitable target for ameliorating the deficiencies in learning and memory associated with Alzheimer's disease (AD). Many ligand-gated ion channel and G-protein coupled receptor systems have been demonstrated to have diminished expression in AD brains. However, GABAA α5 receptor density and function are relatively intact in AD despite evidence for modest reductions in GABAA α5 subunit mRNA.
The simultaneous targeting of the α7 nAChR and GABAA α5 receptors with one molecule is a compelling strategy for the identification of cognition enhancing drugs in neurodegenerative diseases for several important reasons. Activation of α7 nAChRs by agonists, like nicotine, produces selective improvement of working memory. Therefore positive allosteric modulation of the α7 nAChR should also positively impact working memory. α7 nAChRs and GABAA α5 receptors are co-localized to the hippocampus and may promote neurophysiological synergism within the same locale. Negative efficacy modulation of GABAA α5 receptors improves working memory. α7 nAChRs and GABAA α5 receptors are preserved relative to the profound loss of α4β2 nAChRs as AD progresses. Moreover, patent disclosures suggest simultaneously modulating GABA and cholinergic systems with an inverse agonist and agonist, respectively, produces a “ . . . surprisingly effective synergistic combination . . . ” (International published application WO 1999 47142). Multifunctional allosteric modulators of α7 nAChRs and GABAA α5 receptors, such as those embodied in the current disclosure, should mitigate side effects inherent to other potential cholinergic-based therapeutic strategies for cognitive disorders because unlike direct acting α7 nAChR agonists, allosteric modulators will specifically activate the α7 nAChR only in the presence of endogenous agonist (i.e., ACh and choline). Allosteric modulators, in general, do not indiscriminately raise levels of endogenous ACh as with current clinically used acetylcholinesterase inhibitors, such as donepizal.
The allosteric modulators disclosed herein will selectively enhance the sensitivity of α7 nAChRs to the effects of local concentrations of endogenous agonists while preserving the temporal integrity of local neurotransmission. This strategy may be more advantageous than combining two drugs with each particular activity because a molecule with dual sites of action may be synergistic, thus requiring a lower dose than a molecule that targets either site of action alone reducing a) the chances for drug toxicity or b) drug-drug interactions if both receptors were targeted as a drug cocktail.
All references discussed herein are expressly incorporated by reference in their entirety.